ABSTRACT Osteoarthritis (OA) is a leading cause of disability in adults, causing chronic progressive joint pain and tissue damage throughout the joint. No current medical treatments are effective at preventing the progressive joint deterioration, pain and disability characteristic of OA. Although it is known that inflammation and bone- remodeling are major drivers of OA pain and pathology, it is not yet clear which molecular pathways directly drive chronic OA pain and disease progression or are key targets for therapeutic development. Our lab has shown that deficiency of CD14, an inflammatory pattern-recognition receptor expressed by macrophages and other myeloid cell types, protects against OA-associated bone-remodeling and pain-related joint dysfunction after knee injury in mice. In patients, CD14 is increased in joint fluid and associated with intra-articular macrophage infiltration as well as pain severity. This receptor facilitates Toll-like receptor (TLR) signaling. TLRs are innate immune sensors which are important initial triggers of chronic inflammation in response to non-infectious tissue damage. In addition, our team has recently demonstrated that TLR-signaling is critical to development of knee OA pain in mice, via direct stimulation of pain-transmitting (nociceptive) neurons in the dorsal root ganglia (DRG) that innervate the knee. In this proposal, we will test the hypothesis that CD14 on myeloid cells including macrophages promotes OA pain-related pathology (bone remodelling and inflammation), while CD14 also augments OA pain by directly sensitizing neurons innervating the arthritic joint. We will utilize in vitro techniques and the murine destabilization of the medial meniscus (DMM) model of OA, to understand the cellular and molecular mechanisms by which CD14 drives OA pathology and inflammation in joint tissues. Specifically, in Aim 1 we will use a multi-disciplinary approach to determine how genetic deficiency of CD14 modifies inflammation and bone-remodeling during progression of OA, using the DMM model. We will determine the contribution of myeloid cells to inflammation and bone-remodeling in the model by using bone marrow (BM) chimeric mice. Lastly, we will test the effects of CD14 on differentiation of cells that drive bone-remodeling (osteoclasts) and production of inflammatory mediators of pain from myeloid cell types. In Aim 2 we will characterize effects of CD14 on TLR-mediated DRG neuronal activation and joint pain. We will use TLR stimuli with relevance to OA to evaluate DRG responses in vitro, comparing WT and CD14-deficient cells. We will additionally compare pain responses to injection of TLR-stimuli into the joint, and expect that both DRG responses and pain will be blunted in the CD14 deficient strain. Finally, in Aim 3 we will test whether pharmacological targeting of CD14 reduces OA progression and pain after DMM-induced injury. This study will then specify the molecular and cellular framework to design future anti-inflammatory therapeutics for OA aimed at CD14- and TLR-mediated mechanisms.